The objective of the project is to study the determinants of virulence in B. fragilis by: A. defining transferable drug resistance; B. developing a genetic system to obtain mutant lacking suspected virulence factors; and C. developing a phage typing system to investigate the epidemiology of infections caused by B. fragilis. A clindamycin-erythromycin resistance transfer factor has been isolated. It is 10 megadaltons in size as determined by endonuclease restriction analysis and measurement of contour length by electron microscopy. Transfer appears to be by conjugation and it has been shown to occur in vivo in gnotobiotic mice. All tetracycline resistant (tetr) isolates of B. fragilis tested have been demonstrated to transfer tetr when the donor cells are pretreated with tetracycline. We have failed to isolate the tetr transfer factor, despite genetic studies indicating its presence. The presence of a tetr factor has been substantiated by the transformation of an E. coli strain (C-600) to tetr with DNA from B. fragilis. High level penicillin/ampicillin resistance (ampr) has been transferred in B. fragilis. The ampr was mobilized by the tetr transfer factor. A reliable mutagenic system has been developed. Arginine, histidine and methionine requiring auxotrops, lactase and galactose non-fermenting mutants have been isolated. A bacteriophage typing system utilizing 19 phages has been developed which permits the typing of 67% of B. fragilis isolates tested. The past year's studies have given further insight into the sophisticated system of genetic exchanges found in B. fragilis. The replication and expression of genetic material from B. fragilis in E. coli provides additional evidence that there are few barriers for the exchange and expression of DNA in the microbial world and provides a powerful tool to study antibiotic resistance factors from B. fragilis.